PROJECT SUMMARY In this application, we have assembled a multidisciplinary team to execute an integrated mechanistic and translational program to defining neoantigen biology in head and neck squamous cell carcinomas (HNSCC). Despite advances in multimodality treatment, a significant number of HNSCC patients suffer from recurrences and distant metastasis. With recent advances in genomics driven oncology and immunotherapeutics, the outcomes for afflicted HNSCC patients are likely to dramatically improve. However, there is an essential need for a better understanding of HNSCC neoantigen biology as these tumor specific antigens are the final common targets for immunotherapy. To address this key issue in immunotherapeutics, the work we propose will directly address the NIDCR RFA on ?Neoantigen-Based Therapeutic Targeting of Head and Neck Cancers? by completing coupled mechanistic and translational studies. Supporting data provide a strong foundation in which to pursue the proposed work and include intriguing findings regarding neoantigen biology in our high- fidelity mouse models of HNSCC, novel clinical trial specimens in previously untreated patients and immunophenotyping approaches in difficult to treat recurrent and/or metastatic HNSCCs (RM HNSCC). The mouse oral carcinoma (MOC) model that we developed displays strong conservation to human HNSCC in its carcinogen origin, in vivo biology and response to anti-PD1 checkpoint blockade and represents a platform for mechanistic translational studies. Focusing on the MOC22 model, we identified this tumor line to possess both an endogenous retroviral expressed antigen (p15E) and a mutated ICAM1 (mICAM1) gene that yields a neoantigen. Antigen specific T cells for both of these antigens are present in growing MOC22 tumors. Importantly, specific vaccination experiments result in response for both antigens but surprisingly only the mICAM1 neoantigen provided protection from MOC22 tumor growth. These data have important implications for neoantigen biology highlighting the need to not only identity neoantigens but also to define induced responses in T cells. Coupled with these mechanistic approaches, we have initiated a novel neoadjuvant pembrolizumab clinical trial that has shown surprising clinical results and highlights this approach as an ideal setting in which to address neoantigen biology. Finally, we have immunophenotyped RM HNSCC tumor samples towards understanding the low 15-20% response rates to anti-PD1 therapy. These supporting data will be used to interrogate neoantigen mechanistic studies in the MOC model, define neoantigen landscapes in neoadjuvant pembrolizumab treated samples using novel screening methodologies and define single cell RNA- Sequencing transcriptome in antigen specific T cells. Finally, we will further expand on reports of increased mutational burdens in the RM HNSCC setting to define whether this translates into neoantigen reactivity and the associated T cell transcriptome. In conclusion, these Aims together will directly address the goals of this NIDCR RFA that will ultimately provide therapeutic benefit for HNSCC patients.